title: Comparison of membrane targeting strategies for the accumulation of the human immunodeficiency virus p24 protein in transgenic tobacco.
abstract: Membrane anchorage was tested as a strategy to accumulate recombinant proteins in transgenic plants. Transmembrane domains of different lengths and topology were fused to the cytosolic HIV antigen p24, to promote endoplasmic reticulum (ER) residence or traffic to distal compartments of the secretory pathway in transgenic tobacco. Fusions to a domain of the maize seed storage protein γ-zein were also expressed, as a reference strategy that leads to very high stability via the formation of large polymers in the ER lumen. Although all the membrane anchored constructs were less stable compared to the zein fusions, residence at the ER membrane either as a type I fusion (where the p24 sequence is luminal) or a tail-anchored fusion (where the p24 sequence is cytosolic) resulted in much higher stability than delivery to the plasma membrane or intermediate traffic compartments. Delivery to the tonoplast was never observed. The inclusion of a thrombin cleavage site allowed for the quantitative in vitro recovery of p24 from all constructs. These results point to the ER as suitable compartment for the accumulation of membrane-anchored recombinant proteins in plants.
authors.affiliation: Department of Plant Cell Biology, Centre for Organismal Studies, University of Heidelberg, Heidelberg D-69120, Germany. jean-marc.neuhaus@unine.ch.
authors.forename: Goretti
authors.initials: G
authors.lastname: Virgili-López
authors.forename: Markus
authors.initials: M
authors.lastname: Langhans
authors.forename: Julia
authors.initials: J
authors.lastname: Bubeck
authors.forename: Emanuela
authors.initials: E
authors.lastname: Pedrazzini
authors.forename: Guillaume
authors.initials: G
authors.lastname: Gouzerh
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Neuhaus
authors.forename: David G
authors.initials: DG
authors.lastname: Robinson
authors.forename: Alessandro
authors.initials: A
authors.lastname: Vitale
mesh_terms: D000818:Animals; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D016655:HIV Core Protein p24 / Q000096:biosynthesis / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D015028:Zein / Q000096:biosynthesis / Q000235:genetics
chemical_list: D016655:HIV Core Protein p24; D011993:Recombinant Fusion Proteins; D015028:Zein